High Efficiency Transformation

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Day 1
Inoculate the yeast strain into 5 ml of liquid medium and incubate overnight.

Day 2

Determine the titer of the yeast culture by pipetting 10 µl of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10^6 cells/ml will give an OD600 of 0.1.

Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.

Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.

It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.

When the cell titer is at least 2 x 107 cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.

Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.

Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.

Pipette 100 µl samples (ca. 108 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.

Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.

Number of Transformations

Reagents

1

5 (6X)

10 (11X)

PEG 3500 50% w/v

240 µl

1440 µl

2640 µl

LiAc 1.0 M

36 µl

216 µl

396 µl

Boiled SS-carrier DNA

50 µl

300 µl

550 µl

Plasmid DNA plus Water

34 µl

204 µl

374 µl

Total

360 µl

2160 µl

3960 µl

Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.

Incubate the tubes in a 42°C water bath for 40 min.

Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.

Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .

We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants.

Plate appropriate dilutions of the cell suspension onto SC selection medium. For transformation with an integrating plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library plasmid dilute 10 µl of the suspension into 1.0 µl of water and plate 10 and 100 µl samples onto two plates each. The 10 µl samples should be pipetted directly into 100 µl puddles of sterile water on the SC selection medium.